CA2139419C - Purified saccharose synthase, process for its production and its use - Google Patents

Abstract

Nucleotide sugars, especially UDP, ADP, CDP or TDP glucoses can be enzymatically obtained by the reaction of nucleoside diphosphates with di or trisaccharides with a saccharose synthase in which the virtual absence of nucleoside phosphatases (0.1% or less) can be ensured by special purification methods and sensitive detection. The purification of the raw extract, obtained preferably from rice grains, comprises especially the application of the ultra-filtered extract containing 50 mM KCl with a pH 8 on a sepharose Q* column and a gradient elution out of the column at a pH 8 with 50 to 500 mM KCl.

Description

2139~19 PURIFIED SACCHAROSE-SYNTHASE, PROCESS FOR ITS
PRODUCTION AND ITS USE

DESCRIPTION

Sucrose-synthase (Glycosyltransferase EC2.4.1.13 UDPG: D-Fructose-2-glucosyltransferase) is an enzyme which has been long known and is especially widespread in plants (e.g., wheat, rice, corn, sugar beets, etc.) see Y. Milner and others in "Nature" 206 (1965), Page 825; the function of this enzyme as a catalyst in the production of activated sugars in the metabolism of plants has been extensively studied and compendia have been produced (Avigad, G in Loewus, F.A. et al. (eds.) Encyclopedia of Plant Physiology New Series Vol. 13A, Carbohydrates I, Intracellular Carbohydrates, Springer-Verlag, Brelin 1983, pages 217-347). The enzyme catalyzes in vivo the splitting of sucrose according to the following equation:
Sucrose+ NDP ~ ~ NDP-Glucose + Fructose (1) in which N stands for the Nucleoside, like uridine, thymidine, cytidine, guanine and adenine.
Purification and characteristics of the enzyme have been described inter alia by T. Nomura et al in Arch. Biochem. Biophysics 156 (1973), pages 644-652, which gives a yield of 8.8% at 11.4 fold purification by ammonium sulfate precipitation and column chromatography on DEAE-Cell~lose and Neusilin (MgO + Al203 + 2Sio2) and Km-values for synthase reaction and splitting reaction are given.
Recently, S.L. Haynie and G.M. Whitesides have reported in Appl. Biochem. & Biotechnol. 2~ (1990) upon a sucrose-synthesis purified by an ammonium sulfate precipitation based process and its use for sucrose synthesis by reaction and UDP-Glucose and Fructose.

(Replacement Page) ~i3g~19 That describes the limited stability of the enzyme (page 158), especially of highly purified enzyme preparations (page 160) as well as the drawback of highly stabilized enzyme preparations of reduced purity based upon byproduct activity, especially of phosphoglucomutase and the consequent low activity requirement of the use of larger gel volume (of the gel immobilized enzyme).
R.H. Juang and others describe in J. Chinese Biochem. Soc. 17 (1988) 42 - 51. A sucrose-synthase purification by column chromatography and electrophoresis with 38-times purification which is carried out depending upon the protein composition.
In spite of the long known manner functioning of sucrose-synthase and the purification process, the production of activated sugars according to the above equation (1) has not hitherto been economically utilized although the sucrose-synthase is commercially lS available and activated sugars as well as disaccharides and oligosaccharides are of considerable significance in the sugar chemistry.
Mono-,. Oligo- and Polysaccharides have multiple functions as antigen determinants, in cell-cell recognition, in cell differentiation and as binding cites for toxins, bacteria and viruses.
A compilation of the production and use can be found in S.
David and others in Advances in Carbohydrate Chem. A. Biochem. 49 (1991), 175 - 237. Y. Ichikawa and others present in ANal. Biochem.
202 (1992) 215- 238 different reaction mechanisms. As "large scale synthesis", however, especially the reaction of sugar-1-phosphate (especially glucose-1-phosphate) with nucleoside triphosphate, especially with UTP in the presence of pyrophosphorylase, reference may be had to C.H. Wong and others (J. Org. Chem. 47 (1982) 5416 - 18) which describes a multistage enzymatic synthesis of nucleotide sugars.

(Replacement Page) Q
Thls synthesls accordlng to C.H. Wong ls also descrlbed as the method of cholce by Toone and W
hlteslde ln Am. Chem. Soc. Sympos, Sr. 466 (1991) 1 - 22.
It has been surprlslngly found that su crose synthase lsolate (especlally of commerclally avallable en zyme contalned generally more or less hlgh proportlons of nucle otlde phosphatases and that the presence of them, even ln small amounts, ls so greatly detrlmental to the synthe sls of NDP
Glucose and homologous compounds that the conflr med synthesls sultablllty of sucrose synthesls could not be re cognlzed heretofore.
By approprlate purlflcatlon methods an d sensltlve phosphatase determlnatlons, a sucrose synthetase has been developed whlch ls surprlslngly stable and enabl es a clean slngle stage synthesls reactlon accordlng to (1) .
The sub~ect of the lnventlon ls, accor dlngly, a purlfled sucrose synthase whlch ln lts HPLC chro matogram ln whlch ln lts HPLC chromatogram, nucleotlde phosp hatases are no longer detectable (s 0.1 %).
The lnventlon provldes a purlfled sacc harose-synthase that retalns full actlvlty after 5 hour s at 37~C and has a Hlgh Performance Llquld Chromatogram profl le ln whlch nucleotlde phosphatases are not detectable (s 0.
1% w/w).
As sources for the sucrose-synthase, s erve especlally rice, corn or wheat gralns whlch can be sprout whlch have sprouted and are mechanlcally dlslnte grated. The aqueous raw extract thereby recovered ls sub~ect ed elther to a PEG-preclpltatlon (A) or a dlstrlbutlon ln an aq ueous 2-phase 7-~

system ~B). In (A), a fractlonal precipltatlon can be carrled out ln whlch lnltlally ~Al) relatlvely low molecular welght PEG (polyethylene glucose; M> 1000) and reduced PEG
concentratlons are utlllzed for the preclpltatlon of accompanylng protelns (EG wlth 5% PEG 4000) whlle the sucrose synthase remalns ln the supernatant; the latter ls then preclpltated ln a second step (A2) wlth lncreased PEG
concentratlon and from the preclpltate dlssolved out wlth 200 mM Hepes Buffer (pH 7.2). The preclpltatlon (A2) ls not necessarlly requlred and can, or slmpllflcatlon of the process and yleld increase, be omltted as has been lndlcated further below.
The molecular welght of the PEGs ln the PEG-precipitation can be varied with correspondlng change ln the PEG percentage.
The sucrose-synthase whlch ls agaln brought lnto solutlon or the supernatant or the enzyme contalnlng phase of the extractlon ls advantageously after an Adsorption on Sephadex* A50 and stepwise elution at pH 7.2 (set with Hepes-NaOH) with 100 mM KCl and 300 mM KCl and change of buffers as well as ultrafiltration loaded onto a Sepharose*-Q-column and sub~ected to a linear gradlent elutlon wlth 50 - 500 mM KCl at pH 8 (200 mM Hepes-NaOH) and chromatographed on a gel filtration column.
Especially important ls the treatment step on the Sepharose-Q-column wlth gradlent elution as descrlbed. In thls manner, one obtalns purlfled sucrose-synthase whose nucleotlde phosphatase content c 0.1 %, l.e. ln a phosphatase-*Trade-mark - 4 -A

4 ~1 ~
test of the enzyme preparatlon nucleotide phosphatase ls no longer detectable.
The sucrose-synthase purlfled ln accordance wlth the lnventlon permlts use especlally for enzymatlc synthesls of actlvated glucose and actlvated glucose derlvatlves by spllttlng of dlsaccharldes, trlsaccharldes or ollgosaccharldes derlvatlves wlth nucleosldedlphosphates. The resultlng products, for example, UDP-glucose, TDP-glucose and CDP-glucose are lmportant startlng materlals for the enzymatlc and/or chemlcal preparatlons of actlvated desoxy sugars and thelr derlvatlves. A further example of the above-descrlbed use ln the enzymatlc spllttlng of 2-Desoxysaccharose wlth sucrose-synthase wlth the use of Nucleosldedlphosphates, e.g., UDP or TDP.

- 4a -A

2139~I9 In combination with other enzymes, for example, UDP-glucose epimerase and galactosyltransferase, sucrose-synthase can be used for cyclic regeneration of e.g. UDP-glucose. Thus an enzymatic synthesis is carried out of a disaccharide derivative like, for example, N-acetyllactosamine (LacNAc) with three enzymes. In comparison withpublished enzymatic synthesis of LacNAc (Wong and others J. Org. Chem.
47 (1982) 5416 - 5418, an economic advantage is obtained because of reduction in the number of enzymes.
Sucrose-synthase of the invention are purified in accordance with the invention is also usable for the synthesis of glucosides as well as their derivative. Thus UDP-glucose or activated glucose derivatives are transferred to acceptor molecules with at least one hydroxyl group. For example, for sugar molecules as acceptors are for the ketoses the isomers of D-fructose, e.g. D-psicose, D-tagatose and L-sorbose, as well as their derivatives, e.g. 5, 6-didesoxy-5-keto-D-fructose and 6-Desoxy-L-sorbase. Examples of sugar molecules as acceptors as aldoses are L-arabinose, D-lyxose, D-mannose as well their derivatives, e.g. 1,6-Anhydroglucose.
Di-, Tri- and Oligosaccharides are also acceptor molecules, e.g. lactulose, isomaltulose and raffinose. Other hydroxyl group containing acceptor molecules which do not belong to the class of sugars are especially heterocyclic compounds with at least one hydroxyl group on the heterocyclic ring and/or in a side chain found thereon, e.g. l-ethyl-3-hydroxy-pyrollidine or A-(2-hydroxy-ethyl)piperidine.
The process of the invention is described in detail in thefollowing Examples. Reference~is made to the accompanying drawings:
FIG. 1: the chromatogram of the sepharose-Q-separation (Replacement Page) FIG. 2: the NDP-glucose formatlon wlth dlfferent nucleotldes;
FIG. 3 and 4: the formatlon of TDP- and UDP-glucose wlth sucrose-synthase;
FIG. 5 and 6: Reactlon schemes for the enzymatlc synthesls of N-acetyllactosamlne (accordlng to Wong); FIG. 5 or the lnvention; FIG. 6);
FIG. 7 and 8: Nucleotlde-chromatograms of the product mlxture (Step 1 and 2 according to FIG. 6) after the lnactlvatlon of the enzyme;
FIG. 9: The HPLC-chromatogram of the product mlxture (complete cycle accordlng to FIG. 6);
FIG. 10-12: curves of the klnetlcs of the synthesls reactlon (I):
FIG. 13: the lnfluence of metal lons on the enzyme actlvlty; and FIG. 14: a dlagram of the formatlon of TDP-glucose ln the EMR-enzyme membrane reactor (EMR).
Example 1: Isolatlon of the Saccharose-Synthase 800 g of rlce gralns are caused to swell overnlght ln Hepes-NaOH buffer pH 7.2 and are then dlslntegrated ln a Warlng Blender* for 1.5 mlnutes. Thereafter, followlng homogenlzatlon wlth a hand mlxer for a further 3 mlnutes, the pellet ls preclpltated ln a centrlfuge (Sorvall* GS3, 20 mln, 5000 rpm 4~C). Then the proteln ln the supernatant ls preclpltated fractlonally wlth PEG 4000 (5 and 20% PEG). The pellet after the 20% PEG preclpltatlon ls dlssolved ln buffer and bound to Sephadex* A50 ln a batch adsorptlon. 200 ml of *Trade-mark - 6 -A

~ ~ 3 ~ 4 ~
Sephadex-A50-Gel ls charged wlth about 4 g proteln. The stepped elutlon ls begun wlth 300 ml Hepes-NaO~ pH 7.2 and 300 ml Hepes-NaOH pH 7.2 wlth 100 mM KCl. The enzyme ls eluted wlth two volumes (100 ml) Hepes-NaOH pH 7.2 wlth 300 mM KCl.
After reverse bufferlng and ultraflltratlon, thls fraction was charged onto a Sepharose*-Q-column (Hepes-NaOH pH
8.0 wlth 50 mM KCl) and eluted wlth a llnear gradlent (50 mM -500 mM KCl ln Hepes-NaOH 200 mM pH 8.0) the collected enzyme fractlon ls flnally chromatographed ln a gel flltratlon column (Superdex* 200 prep grade).
Sucrose-synthase from the rlce ls enrlched 151 tlmes wlth the yleld of 5.4% (Table lA). A greater loss ls obtalned by the preclpltatlon wlth polyethyleneglycol 4000 (about 80%
loss wlth 5 to 20% preclpltation). Alternatlvely, an enzyme enrlchment can be provlded of the raw extract wlth the ald of an aqueous 2-phase system (PEG/Salt). Very effectlve are the batch adsorptlon on Sephadex* A50 and the subsequent anlonlc chromatography on Sepharose*-Q-fastflow wlth a purlflcatlon factor of 43 ln toto. Aslde from thls, the nucleotlde dlsphosphatase and monophosphatase actlvlty ls completely separated out (FIG. 1) whlch ls very lmportant for the use of the sucrose-synthase ln enzymatlc synthesls.
The molecular welght of the natlve enzyme amounted to 362,000 ~ 7,000 Da, lt ls comprlsed of 4 subunlts each of 90,000 Da and has no lntermolecular or lntramolecular disulfide brldges. The N-termlnal amlnoacld sequenclng by an automated Edmand decomposltlon ln a pulse llquld proteln sequencer lndlcated that the N-termlnal of the subunlt ls *Trade-mark - 7 -A

blocked. The lsoelectrlc polnt of the natlve enzyme was at pH
6.16.
The purlflcatlon of the saccharose-synthase was optlmlzed based upon the startlng amount of rlce and the number of purlflcatlon steps. Table lB lndlcates that the yleld wlth only sllghtly reduced purlty could be lncreased to 21%. The purlflcatlon encompassed lnstead of 6 only 4 steps, whereby the preclpltatlon wlth 20% PEG 4000 and the batch adsorption on Sephadex A50 were omltted. The purlfled enzyme ls also free from nucleotlde monophosphatases and nucleotlde dlsphosphatases also after thls purlflcation.
TABLE 1 A:

Method Volume Proteln Actlvlty Yleld Purlflcation ml] [mg] [U] [%] Factor -Tlmes Dlslntegratlon 704 5140 30.4 100 5 - 20 % (w/w)40 632 5.1 17 1.4 Sephadex* A50250 135 8.9 29 ll Sepharose* Q 80 17.6 4.5 15 43 Superdex* 20018 1.8 1.6 5.4 154 *Trade-mark - 8 -; 70577-85 A

Method Volume Proteln Actlvlty Yleld Purlflcatlon [ml] [mg] [U] [%] Factor -Tlmes Dlslntegratlon 4555 15944 113.4 100 SUPERNATANT
5 % (w~w) 4500 10350 76.1 67 1.03 Sepharose* Q1300 1950 66.4 58.6 4.8 Superdex* 20080 28.8 24.3 21.4 118.9 *Trade-mark - 9 -A

The investigation of the splitting reaction and synthesis reaction with sucrose synthase has indicated that 1. Sucrose-Synthase is suitable for the enzymatic synthesis of UDP-glucose, TDP-glucose, GDP-glucose and CDP-glucose from sucrose.

2. The combination of sucrose-synthase with other enzymes (see above) can be used for the enzymatic synthesis of secondary nucleotide sugars (UDP-galactose, UDP-glucouronic acid).

3~ In the enzymatic synthesis of Oligosaccharides in the enzyme membrane reactor the use of sucrose-synthase for "cofactor regeneration" represents a significant simplification of the kinetic control.

4. The substrate spectrum of sucrose-synthase for Di-Tri- and Oligosaccharide as well as glucoside gives rise to hitherto not accessible activated Mono-, Di- and Oligosaccharides.

5. Other nucleotide sugars than UDP-glucose can be used in the synthesis reaction with fructose.

6. Fructose can be replaced with other sugars with ~-furanose configuration as well as by sugar alcohols and other chemical compounds with structural similarity to ~-furanose.

Below examples of the mode of action and use of the saccharose-synthase are described:

1. Substrate spectrum of the nucleosidediphosphase.
UDP, TDP, CDP, ADP and GDP were investigated. The reaction compositions contained:
550 ~1 Hepes-NaOH (200mM, pH 7.2) 250 ~1 Sucrose (2 M) 100 ~1 Nucleosidediphosphate (15 - 90 mM) 100 ~1 purified Sucrose-synthase t21.1 mU/ml) (Replacement Page 2139~19 The reaction composition was incubated at 30 C and stopped at different times (5 min. at 95~C). After filtration of the sample through a 0.22 ~m filter, the resulting nucleotide sugar was analyzed by means of ionpair HPLC.
The formation of UDP-glucose and TDP-glucose was quantified based upon calibration curves for the HPLC chromatogram (peak area/concentration).
FIG. 2 shows that the nucleosidediphosphate was accepted in the sequence UDP, TDP, ADP, CDP and GDP. The purified enzyme was free from nucleotidephosphatases (NPases, control without sucrose), which decompose the nucleosidediphosphates to the monophosphates or also to their bases. In the HPLC chromatogram, the peaks which arise for UMP, TMP, uridine and thymidine were recycled by suitable control test upon impurities on the substrate and by the decomposition of the nucleosidediphosphate by the heat treatment. After heat treatment of 1.57 mM UDP, 0.123 mM UMP results; of that 0.082 mM (5%) was already present as impurities in the UDP substrate.
From an investigation into the concentration characteristic with time of the syntheses of UDP-glucose and TDP-glucose, it is clear that with increasing quantities of enzymes the practically complete reaction of the nucleosidediphosphate to nucleotide sugar can be achieved in short reaction times.
Table 2 shows the reaction rates for the syntheses of UDP-and TDP-glucose together. With 0.16 mU enzyme, the space-time yield increases only slightly with higher substrate concentrations. With -1.8 mU sucrose-synthase, after 3 hours, a 99% reaction calculated on the UDP concentration (1.57 mMr at starting can be achieved. This corresponded to a space-time yield of 0.21 g/l h. With a higher UDP
concentration (1.86 mM), the space time yield amounted to 0.4 g/l h.

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21394~

The space time yield for the synthese of TDP-glucose amounted to 0.011 g/l h for 0.16 mU of enzyme at start; the conversion corresponded to 49 % after 30 h for 0.16 mU enzyme. A further experiment indicated that the conversion of 8/2 mM TDP with 0.2 mU
enzyme after 24 hours was increased to 80%.
For comparison, a commercial preparation of the saccharose-synthese from wheat was tested ~specific activity 8.15 mU/mG). This enzyme showed similar space-time yields as the purified enzyme from rice. However, the HPLC chromatogram indicated the simultaneous formation of relatively large amounts of UMP and uridine through Nucleotidephosphatases (Table 2). These enzyme impurities are not present in purified enzyme from rice. The resulting UMP- and uridine peaks in the chromatogram can be recycled only by heating the specimens.

2. Buffer Spectrum The buffer spectrum for the syntheses of UDP- and TDP-glucose was investigated with the purified sucrose-synthase from rice and the commercial enzyme from wheat. The following buffers were tested (all at 200 mM and pH 7.2, the pH buffer ranges are given):

Mops-NaOH-NaCl pH 6.25 - 8.15 TES-NaOH-NaCl pH 6.55 - 8.45 Tris-HCl pH 7.00 - 9.00 Hepes-NaOH pH 6.80 - 8.20 KH2PO4-NaOH pH 5.80 - 8.00 Na2HPO~-NaH2Po4 p~ 5.80 - 8.00 Imidazole pH 6.20 - 7.80 TEA-Hydrochloride - NaOH pH 6.80 - 8.80 The Incubation composition contained:

(Replace~ent Page) 2139~19 550 ~1 ~uffer (200 mM, pH 7.2) 250 ~1 Sucrose (2 M) 100 ~1 UDP (15.7 mM) or TDP (16.4 mM) 100 ~1 Enzyme solution Incubation and Analysis were carried out as described under 1.
It was found that the hitherto used hepes-NaOH-buffer was most suitable for the synthesis of UDP-glucose and TDP-glucose. For both enzymes, the Mops-buffer and the TES-buffer gave 60 to 80~
residual activity. The commercial enzyme indicated, by contrast to the rice enzyme, in TEA-buffer, an about 30 to 40 ~ higher residual activity. In the remaining buffers, both enzymes had a residual activity of less than 50%.
These results indicate that the selection of the buffers is significant to the activity of the sucrose-synthase and the lS determination of the pH optimum can be influenced thereby.

3. pH-Optimum The following buffers were used for the determination of the pH optimum (all at 200 mM):
Na-Citrate-Citric acid pH 4.0 - 6.2 KH2PO~-NaOH pH 5.8 - 7.2 Mops-NaOH-NaCl pH 6.3 - 7.4 Hepes-NaOH pH 6.8 - 8.2 TEA-hydrochloride - NaOH pH 7.2 - 8.8 The incubation composition contained:
640 ~1 buffer (200 mM) 250 ~1 Sucrose (2 M) 100 ~1 UDP (15.7 mM) or TDP (16.4 mM) 10 ~1 Enzyme solution (Replacement Page) Incubation and analysis are affected as described under 1.
Both enzymes show different pH optima independence upon the buffer used.
With UDP as the substrate, both enzymes have a pH optimum between 5.5 and 5.7 with use of citrate buffer or phosphate buffer.
With use of the Hepes buffer and Mops buffer, the optimum for the UDP-glucose synthesis lies between pH 6.7 and 7Ø
For the synthesis of TDP-glucose this is in the same way;
with citrate buffer or phosphate buffer the optimum lies between pH
5.8 and 6.2 and with Mops buffer or Hepes buffer between 6.5 and 6.8.

4. pH Stability For determining the pH stability, the rice enzyme is incubated at different pH values in Hepes-NaOH-buffer (200 mM) and for different space-temperatures of different durations. The enzyme is then subjected to the usual activity test:

550 ~1 Hepes-NaOH (200 mM, different pH values) 250 ~1 Sucrose (2 M) 100 ~1 UDP (20 mM) 100 ~1 Enzyme solution After lh reaction time, the specimens were analyzed as described above with HPLC.
The purified sucrose-synthase from rice shows at pH 7.0 and 7.9 after 2 hours a residual activity of > 60%, which allows its use for the synthesis of UDP-glucose and TDP-glucose.
.

(Replacement Page) 2~3~41~

5. Temperature Optimum For determining the temperature optimum, the rice enzyme was incubated at pH 6.5 tpH optimum) and different temperatures.
Thereafter, the enzyme was subjected to the following activity test:

550 ~l Buffer (200 mM, pH 6.5) 250 ~l Saccharose (2 M) 100 ~l UDP (20 mM) 100 ~1 Enzyme solution.
After 1 hour of reaction time, the samples were analyzed as described above with HP~C. Additionally, a respective control for each was incubated without enzymes and analyzed.
For the splitting of sucrose with UDP, the temperature optimum of the sucrose-synthase from rice at pH 6.5 was between 50~
and 60 C.

6. Temperature stability The enzyme retained after 5 hours at 37~C its full activity, after 5 hours at 56 C a residual activity of 37% was present.

8. Kinetics To determine Vm~ and ~ of the substrate, UDP or TDP was varied with constant sucrose concentration from 500 mM between 0 and 10 mM in the reaction mixture. With a constant UDP (2mM) concentration, the sucrose was~varied in the reaction composition between 0 and 500 mM. All reaction mixtures were incubated for lh at 30 C and pH 6.5 (Hepes-NaOH 200 mM). The samples (Replacement Page) 2139~19 were treated as described above and analyzed with HPLC. The rice enzyme indicated for UDP a substrate excess inhibition (Ki(S) = 16 mM
with 0.9 mU enzyme) at a Km-value of 0.4 mM (FIG. 10). The Km-value for TDP amounted to 0.65 mM (FIG. 11). The substrate excess inhibition can be countered by higher enzyme quantities. The Km-value for saccharose amounted to 108 mM (FIG. 12).

9. Dependence on Divalent Metallions FIG. 13 shows that in the presence of 1 mM of metallions, for example Mn2+ and Mg2t the activity of the saccharose synthase is influenced only slightly. A stimulation of the enzyme activity occurred with Mn2t and Ca2t with TDP as the substrate. In the presence of CU2~ and Fe2t the enzyme is completely inactivated.

10. Enzymatic Synthesis of UDP-glucose and TDP-glucose under Optimum Conditions The reaction composition contained:
550 ~1 Hepes-NaOH t200 mM, pH 7.2) 250 ~1 Saccharose (2 M) 100 ~1 Nucleosidediphosphate (UDP 100 mM or TDP 124 mM) 100 ~1 Purified sucrose-synthase (15 mU/ml) The reaction composition was incubated with UDP at pH 7.0 or with TDP at pH 6.~ at 30~C and stopped at various times (5 min at 95~C). After filtering the specimens through a 0.22 ~m filter, the resulting nucleotide sugar was analyzed by means of ionpair HPLC.
The formation of UDP-glucose and TDP-glucose was quantified with calibration curves for the HPLC chromatogram tpeak area/concentration).

(Replacement Page) 21~9419 FIGS. 3 and 4 show that after 24 h 92% of the UDP is converted to UDP-glucose and 84% of the TDP to TDP-glucose.
The purified sucrose-synthase was introduced into an enzyme membrane reactor (10 ml reactor-volume) for enzymatic synthesis of TDP-glucose. FIG. 14 shows the conversion of TDP to TDP-glucose and the concentrations of TDP, TDP-glucose and fructose at 5 mM TDP, 350 mM saccharose, 40 minutes residence time and 990 mU sucrose-synthase The conversion amounted to 89.6 % calculated on the TDP introduced.
The theoretical space time yield for a one-liter reactor volume gave 98.1 g TDP-glucose per liter and day.

ll. Substrate Spectrum of the Sucrose-Synthase for the splitting reaction (I) For the splitting reaction of the sucrose-synthase, sucrose was replaced by other disaccharide or trisaccharide:
~-Glc 1-2 ~-Fruc + UDP (TDP) UDP-Glc (TDP-Glc) The concentration of the saccharide amounted to 75 to 500 mM
in the reaction composition. 2 mM UDP or TDP and as a rule 10 to 80 mU enzyme were introduced. After 3h at 30 C and pH 7.2 (200 mM Hepes-NaOH), the reaction was stopped at 95~C 5 min. The formation of nucleotide sugars was followed with HPLC and compared with a control (without enzyme).
Table 3 shows that the disaccharide isomaltulose (Palatinose) and the trisaccharide raffinose and melezitose can replace the saccharose.

(Replacement Page) 12. Substrate Spectrum of the Sucrose-synthase from rice for the synthesis of Disaccharides.

A: Variation of the Nucleotide Sugar.
For the synthesis reaction the nucleotide sugars were varied:
UDP-Glucose + Fructose Saccharose + UDP

Initially only UDP activated sugar was introduced:
UDP-galactose, UDP-N-acetylglucosamine, UDP-glucouronic acid, UDP-N-acetylgalactosamine.
The composition contained:

550 ~1 Buffer ~200 mM, pH 7.5) 250 ~1 Fructose (40 M) 100 ~l UDP-Sugar t20 mM) 100 ~l Enzyme Solution The composition was incubated for 2 hours at 30~C and 15 stopped for S minutes at 95 C. With HPLC, with comparison with a control twithout enzyme) the development of UDP was followed.
Apart from UDP-glucose, UDP-N-acetylglucosamine and UDP-xylose can be reacted with the rice enzyme. (Table 4).

tRePlacement Page) TABLE 3: TEST OF THE SUBSTRATE SPLITTING WITH PURIFIED SUCROSE-SYNTHASE FROM RICE AND UDP FOR SYNT~ESIS OF ACTIVATED SUGARS
Name Linkage Relative Activity Saccharose ~ Glc 1-2 ~ Fruc 100 Saccharose-6'-P
2-deoxy-saccharose 55 Turanose ~ Glc 1-3 ~ Fruc Isomaltulose ~ Glc 1-6 ~ Fruc 1.0 Lactulose ~ Gal 1-4 Fruc Trehalose ~ Glc 1-1 ~ Glc Maltose ~ Glc 1-4 Glc Isomaltose ~ Glc 1-6 Glc Laminaribose ~ Glc 1-3 Glc Cellobiose ~ Glc 1-4 Glc ~-gentiobiose ~ Glc 1-6 Glc Mannobiose ~ Man 1-3 Man N'N'Diacetyl- ~ GlcNAc 1-4-chitobiose GlcNAc ~-lactose ~ Gal 1-4 ~ Glc ~-lactose ~ Gal 1-4 ~ Glc ~-D-melibiose ~ Gal 1-6 Glc LacNAc ~ Gal 1-4 GlcNAc Ampicillin 2.4 Chlorogenic acid Thiodigalactoside Thiodiglucoside p-aminophenyl-~-L-fucopyranoside 3-0-~-D-galacto-pyranosyl-D-arabinose Octyl-~-D-glucopyranoside p-aminophenyl-~-D-galactopyranoside Raffinose ~ Gal 1-6 ~ Glc 1-2 ~ Fruc 3.8 Melizitose ~ Gal 1-3 ~ Fruc ~ Glc 0.4 (Replacement Page) 2139~19 TABLE 4: SUBSTRATE SPECTRUM OF THE PURIFIED SACCHAROSE SYNTHASE FROM
RICE DIFFERENT UDP-SUGARS WERE USED WITH FRUCTOSE AS ACCEPTOR

Name Relative Activity %

UDP-glucose 100 UDP-galactose UDP-N-acetylglucosamine 1.8 UDP-N-acetylgalaktosamine UDP-glucuronic Acid UDP-xylose 1.7 12. Substrate Spectrum of the Saccharose-synthase from rice for the synthesis of Disaccharides.

B: Variation of the Acceptor.
For the synthesis reaction, the acceptor was varied. Apart from the natural acceptor, other diastereomers of D-fructose, like D-psicose, D-tagatose, D-sorbose were introduced. Further, several keytoses were systematically tested. Apart from Aldoses, nonsugar acceptors were also tested.
Table S indicates that the tested Diastereomers of D-fructose except from D-sorbose all are acceptors. L-sorbose, D-xylulose and the deoxyketoses are also acceptors of sucrose-synthase.
Of the aldoses, D-mannose, D-lyxose and L-arabinose were acceptors.
As acceptors, derivatives of glucose are available, e.g. 1,6 anhydro-~-D-glucose or octyl-~-D-glucopyranoside.
Disaccharide and trisaccharide (e.g. lactulose or raffinose) can also serve as acceptors. Of the nonsugar acceptors, derivatives of pyrrolidine can be introduced into the synthesis reaction of the sucrose synthase.

~Replacement Page) TABLE 5: SUBSTRATE SPECTRUM OF THE PURIFIED SUCROSE-SYNTHASE FROM
RICE
VARIOUS ACCEPTOR SUBSTRATES WERE REACTED WITH UDP-GLUCOSE
Name Relative Activity %
D-fructose 100 Sedoheptulose Anhydride 1.1 Mannoheptulose D-psicose 14.1 D-tagatose 28.3 D-sorbose L-sorbose 6.0 D-ribulose D-xylulose 40.7 L-xylulose D-erythrulose 5-keto-6-deoxy-D-fructose 25.1 5,6-dideoxy-5-methyl-D-fructose19.0 5,6-dideoxy-D-fructose 7.6 6-desoxy-L-sorbase 8.8 D-glucoheptose ~-D-allose ~-L-allose D-altrose D-glucose L-glucose D-mannose ~ . 3.9 L-mannose - - 30 L-gulose D-idose L-idose D-galactose L-galactose ~-D-talose D-ribose tReplacement Page) 2139~19 Name Relative Activity %
L-ribose D-arabinose L-arabinose 3.2 D-xylose L-xylose D-lyxose 14.7 D-sorbitol D-arabitol L-arabitol L-ascorbic acid 1,6-Anhydroglucose 9.1 n-octyl-~-D-glucopyranoside 0.5 Hydroxypyruvate 3-Hydroxybenzaldehyde 3-Hydroxy-tetrahydrofuran Tetrahydro-3-furan-methano1 S(+)-2-(hydroxymethyl)-pyrrolidine 3-hydroxypyrrolidine 6.3 3-hydroxy-N-methyl-pyrrolidine 1.9 l-ethyl-3-hydroxy-pyrrolidine 10.4 3-pyrrolidino-1,2-propandiol 9.6 N-(2-hydroxymethyl)piperidin 9.4 Tropin . 10.3 Turanose 0.5 Lactulose 12.3 Raffinose 8.4 Isomaltulose 3.1 ~-lactose Melizitose 0.6 (Replacement Page) ~139419 13. ENZYMATIC SYNTHESIS OF UDP-GALACTOSE AND N-ACETYLLACTOSAMINE
ACCORDING TO THE DIAGRAM OF FIG. 6 UDP-galactose and N-acetyllactosamine were produced according to FIG. 6 (FIG. 5 shows the synthesis cycle according to WONG) Composition:

677 ~l Hepes-buffer (50 mM, pH 7) 100 ~l UDP (100 mM) 100 ~l Saccharose 123 ~l Enzyme (10 mU in composition) After 3 hours of incubation at 30~C, 80% of the UDP was converted to UDP-glucose as determined by HPLC analysis. Thereafter, 100 mU UDP-galactose-epimerase was added and incubated at 30 C
overnight. FIG. 7 shows that UDP-galactose results from UDP-glucose.
Since the equilibrium of the UDP-galactose-epimerase lies strongly on the side of UDP-glucose, UDP-glucose/UDP-galactose ratios of 0.3 are expected (see the peak height ratios of UDP-glucose/UDP-galactose in FIG. 7).
UDP-galactose can also be enzymatically produced by simultaneously incubating the requisite enzyme in the composition.

Composition:

500 mM Sucrose 1-10 mM UDP
3-30 mU Sucrose-synthase 200 mU UDP-gal-epimerase All in 200 mM Hepes-buffer pH 7.2 The results are documented in FIG. 8.

~eplacement Page) For enzymatic synthesis of N-acetyllactosamine, the following test was carried out.

1 mM UDP
1 mM MnCl2 5 mM N-acetylglucosamine 500 mM saccharose 200 mU UDP-gal-epimerase 100 mU ~-1,4-galactosyltransfer 120 mU sucrose-synthase All in 200 mM Hepes-NaOH-buffer pH 7.2 at 30~C overnight.

FIG. 9 shows that N-acetyllactosamine is formed with the aid of the three enzymes (FIG. 6). The conversion amounted to 80% based upon the starting concentration of N-acetylglucosamine.

In the previous tests, sucrose-synthase isolated from rice grains were used; however sucrose-synthase obtained from wheat, interalia, observing the requirements of the invention and usable for single stage formation of activated monosaccharide can be employed as well.

(Replacement Page)

Claims (17)

THE EMBODIMENTS OF THE INVENTION IN WHICH AN EXCLUSIVE
PROPERTY OR PRIVILEGE IS CLAIMED ARE DEFINED AS FOLLOWS:

1. A purified saccharose-synthase that retains full activity after 5 hours at 37°C and has a High Performance Liquid Chromatogram profile in which nucleotide phosphatases are not detectable (~ 0.1% w/w).

2. A purified saccharose-synthase according to claim 1 wherein 1,6-anhydroglucose and 3-hydroxypyrrolidine are among acceptor substrates of USP-glucose.

3. A purified saccharose-synthase according to claim 1 or claim 2 extractable from plant material.

4. A purified saccharose-synthase according to claim 3 wherein said plant material comprises rice grain.

5. A process for preparing a saccharose-synthase having a High Performance Liquid Chromatogram profile in which nucleotide phosphatases are not detectable (~ 0.1% w/w) which process comprises subjecting a saccharose-synthase-containing extract of plant origin to gradient elution from an agarose column at a pH of about 8 with from about 50 to about 500 mM
KCl to yield said saccharose-synthase.

6. A process according to claim 5 wherein said agarose column comprises a Sepharose*-Q-column.

7. A process according to claim 5 or claim 6 wherein said saccharose-synthase is subsequently chromatographed in a gel filtration column.

8. A process according to claim 7 wherein said gel filtration column comprises Superdex* 200.

9. A process according to claim 5 or claim 6 wherein said extract is ultrafiltrated and adjusted to a pH of about 8 and a KCl concentration of about 50 mM prior to gradient elution.

10. A process according to claim 9 wherein prior to ultrafiltration and pH adjustment said extract has been subjected to polyethylene glycol precipitation to remove non-saccharose synthase proteins or to aqueous 2-phase separation.

11. A process according to claim 10 wherein said saccharose-synthase is precipitated by polyethylene glycol and subsequently dissolved in buffer prior to ultrafiltration.

12. Use of a saccharose-synthase according to any one of claims 1 to 4 for reacting a nucleoside diphosphate with a disaccharide, a trisaccharide or an oligosaccharide or a derivative thereof to form a nucleotide sugar or nucleotide sugar derivative.

13. Use according to claim 12 wherein said nucleoside diphosphate is uridine diphosphate, adenosine diphosphate, thymidine diphosphate or cytidine diphosphate and said disaccharide in sucrose.

14. Use according to claim 12 of a saccharose synthase together with an epimerase or transferase to prepare an activated sugar or sugar derivative with a modified sugar group or to transfer a sugar to an acceptor molecule.

15. Use according to claim 14 wherein said acceptor molecule is a sugar.

16. Use of a saccharose-synthase according to any one of claims 1 to 4 to transform activated glucose or a derivative thereof with a hydroxyl group containing acceptor to form a glucoside or a derivative thereof.

17. Use according to claim 16 wherein said acceptor is a sugar.